Our quantitative characterization of highly synchronous infection by the autonomous parvovirus minute virus of mice (MVM) has led to the identification of a number of areas of regulation of the viral life cycle that had been previously unidentified. Two of the most striking of these, transcriptional transactivation of the viral capsid-gene P38 promoter, and the efficient translation of viral mRNA during infection, are directly controlled by the viral nonstructural proteins NS1 and NS2, respectively, and are the object of this proposal. We will examine NS1 transactivation by: A) performing a detailed functional analysis of the role of NS1 in transactivation of the minimum transactivatable P38 promoter unit; and B) by characterizing the higher-- order regulation imposed on P38 in the context of the complete viral genome. We will further characterize the role of NS2 in the translation of viral mRNA during infection by: A) performing a structure/function analysis of NS2 using multiple NS2 mutants; B) determining why NS2's role in facilitating the efficient translation of viral mRNA is only required during infection; C) determining the cis-acting element(s) within the MVM VP2-encoding R3 mRNA that confers NS2 responsiveness, and the nature of its interaction with NS2; and D) determining the point at which viral mRNA translation is blocked in cells infected by NS2 mutants, using biochemical characterizations. Successful completion of these experiments will provide information critical to our understanding of how parvoviruses replicate during acute infection and maintain their presence in animal populations. In addition, the detailed emerging picture of the virus life cycle demonstrates that both the regulatory mechanisms that control parvovirus gene expression and the nonstructural proteins that are involved, have features unique amongst viruses of eucaryotic cells, and so understanding of these regulatory mechanisms and participating proteins will also provide a deeper insight into cellular process of gene expression.